Elucidating microRNA-34a organisation within human Argonaute-2 by dynamic nuclear polarisation-enhanced magic angle...

Area of Science:

• Molecular Biology
• Structural Biology
• Biophysics

Background:

• MicroRNA (miRNA) regulation of gene expression is crucial for cellular processes.
• The RNA-induced silencing complex (RISC), particularly human Argonaute-2 (hAgo2), is central to miRNA function.
• Dysregulation of miR-34a is implicated in various cancers, highlighting its importance.

Purpose of the Study:

• To elucidate the structural organization of miR-34a within hAgo2.
• To investigate the structural changes of miR-34a upon binding to hAgo2 and SIRT1 mRNA.
• To understand the molecular dynamics of miRNA-mediated mRNA regulation.

Main Methods:

• Guanosine-specific isotopic labeling.
• Dynamic Nuclear Polarization (DNP)-enhanced Magic Angle Spinning (MAS) Nuclear Magnetic Resonance (NMR).
• Homonuclear correlation experiments and C8-C1' correlation analysis.

Main Results:

• miR-34a adopts an increased non-A-form helical conformation within hAgo2 and when bound to SIRT1 mRNA.
• Nucleotide-specific sugar puckering (C2'-endo and C3'-endo) distributions were identified.
• Diverse dynamic conformations of miR-34a were captured at cryogenic temperatures (90 K).

Conclusions:

• Cryogenic DNP-enhanced MAS NMR can capture dynamic states of RNA-protein complexes.
• The structural dynamics of miR-34a within hAgo2 are critical for mRNA regulation.
• These findings provide molecular insights into miRNA-mediated gene silencing mechanisms.